Antiviral prodrugs and nanoformulations thereof

ABSTRACT

The present invention provides prodrugs and methods of use thereof.

CROSS REFERENCE

This application is a national entry under 35 U.S.C. § 371 ofInternational Application No. PCT/US2019/063498, filed Nov. 28, 2019,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 62/772,852, filed Nov. 29, 2018. The foregoingapplications are incorporated by reference herein.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grants Nos. R01MH104147, P01 DA028555, R01 NS036126, P01 NS031492, R01 NS034239, P01MH064570, P30 MH062261, P30 AI078498, R01 AG043540, R56 AI138613, andR01 AI158160 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to the delivery of therapeutics.More specifically, the present invention relates to compositions andmethods for the delivery of therapeutic agents to a patient for thetreatment of a disease or disorder.

BACKGROUND OF THE INVENTION

Remarkable progress has been made in the development of effectivediagnostics and treatments against human immunodeficiency virus type one(HIV-1). Antiretroviral therapy (ART) has markedly reduceddisease-associated morbidities and mortality, enabling a nearly normalquality of life for infected people (Vittinghoff, et al. (1999) J.Infect Dis., 179(3):717-720; Lewden, et al. (2007) J. Acquir. ImmuneDefic. Syndr., 46(1):72-77). However, ART requires life-long treatmentin order to suppress viral replication and prevent AIDS onset. Moreover,the effectiveness of ART can be hampered by HIV-1 resistance, drugtoxicities, and poor patient adherence (Wensing, et al. (2014) Top.Antivir. Med., 22(3):642-650; Siliciano, et al. (2013) Curr. Opin.Virol., 3(5):487-494; Prosperi, et al. (2012) BMC Infect. Dis.,12:296-296; Van den Berk, et al. (2016) Abstract number: 948, InConference on Retroviruses and Opportunistic Infections, 22-25;Siefried, et al. (2017) PLoS One 12(4):e0174613). Treatment fatigue,lack of financial and social support, co-existing mental symptoms,and/or substance abuse can result in the failure to adhere to criticalART regimens (Tucker, et al. (2017) EBioMedicine, 17:163-171).

It is now well accepted that long-acting antiretroviral drugs (ARVs) canreduce viral transmission, prevent new infection, affect regimenadherence and limit to the emergence of viral drug resistance andsystemic toxicities (Spreen, et al. (2013) Curr. Opin. HIV AIDS8:565-71; Williams, et al. (2013) Nanomedicine (Lond) 8:1807-13).Poloxamer-coated nanocrystals were made from insoluble compounds as longacting parenterals (LAP) (Rabinow, B. E. (2004) Nat. Rev. Drug Discov.,3:785-96). Long-acting parenteral (LAP) antiretroviral drugs haveimproved regimen adherence (Spreen, et al. (2013) Curr. Opin. HIV AIDS,8(6):565-571). Reducing the treatment schedule from daily to monthly oreven less-frequent administration provides greater patient privacy andsatisfaction and improves regimen adherence (Sangaramoorthy, et al.(2017) J. Assoc. Nurses AIDS Care, 28(4):518-531; Carrasco, et al.(2017) Afr. J. AIDS Res. 16(1):11-18; Williams, et al. (2013) Nanomed.Lond. 8(11):1807-1813). However, only a few antiretroviral drugs havebeen successfully reformulated into LAPs.

The most notable ARV LAPs are cabotegravir (CAB) and rilpivirine (RPV),which when administered once/month can elicit comparable antiretroviralactivity to daily oral three-drug combinations for maintenance therapy(Margolis, et al. (2017) Lancet 390:1499-510). However, there arelimitations to any widespread use of CAB RPV LAP combinations as theyrequire large injection volumes, show injection site reactions withlimited drug access to infectious reservoirs (Zhou, et al. (2018)Biomaterials 151:53-65; Margolis, et al. (2017) Lancet 390:1499-510;Markowitz, et al. (2017) Lancet HIV 4:e331-e40).

CAB is an integrase inhibitor or integrase strand transfer inhibitor(INSTI) with low aqueous solubility, high melting point, high potency,long half-life, and slow metabolic clearance (Karmon, et al. (2015) J.Acquir. Immune Defic. Syndr., 68(3):39-41; Trezza, et al. (2015) Curr.Opin. HIV AIDS 10(4):239-245). These properties enable CAB to beformulated in a 200-mg/mL suspension (CAB LAP) and administeredintramuscularly monthly or even less frequently (Margolis, et al. (2017)Lancet 390(10101):1499-1510; Spreen, W. W. (2014) J. Acquir. ImmuneDefic. Syndr., 67(5):481-486). Notably, CAB plus rilpivirine (RPV) isthe first long-acting combination ART regimen where monthly or everyother month CAB and RPV LAP formulations have demonstrated comparableantiretroviral activity to daily oral three-drug combinations formaintenance therapy (Margolis, et al. (2017) Lancet390(10101):1499-1510).

However, the dosing pattern of CAB LAP has limitations. Specifically,split injections given in 2 mL volumes are required which leads totreatment cessations because of intolerable injection site reactions(Margolis, et al. (2017) Lancet 390(10101):1499-510; Markowitz, et al.(2017) Lancet HIV 4(8):331-340). Moreover, the maximal dosing intervalis only 8 weeks. Recently, the administration of CAB LAP every 12 weekshas been tested with the aim of maintaining plasma CAB concentrationsabove 4 times protein-binding-adjusted 90% inhibitory concentration(4×PA-IC₉₀, 660 ng/mL), a concentration demonstrated to be protectiveagainst new infections in macaques (Spreen, W. W. (2014) J. Acquir.Immune Defic. Syndr., 67(5):481-486; Andrews, et al. (2014) Science343(6175):1151-1154; Andrews, et al. (2015) Sci. Transl. Med., 7(270)270ra4; Radzio, et al. (2015) Sci. Transl. Med., 7(270) 270ra5-270ra5;Andrews, et al. (2016) AIDS 2016:461-467; Markowitz, et al. (2017)Lancet HIV 4(8):331-340; Spreen, et al. (2014) J. Acquir. Immune Defic.Syndr., 67(5):487-492). However, two-thirds of participants had fasterthan anticipated drug absorption leading to plasma drug concentrationsbelow the targeted effective concentration of 4×PA-IC₉₀ at 12 weeks.Thus, ways to extend the dose interval beyond 8 weeks and reduceinjection volumes to improve regimen adherence are greatly needed (Boyd,et al. (2017) Lancet 390(10101):1468-1470).

Complementary to LAP ARVs are preclinical implantable devices for longersustained release. Each can facilitate the other with a noted exception.Indeed, the implantable devices require professional insertion andmonitoring. Further, limitations exist in scale up and processingincluding a potential for “drug dumping” from biodegradable polymers. Inaddition, toxicity from organic solvents and high gel viscosity canprovide local irritation (Kranz, et al. (2001) Int. J. Pharm.,212:11-8). While biodegradable implants provide some additionalbenefits, removals for adverse events or local trauma remainschallenging.

In view of the foregoing, it is clear that improved long term deliveryof ART is needed.

SUMMARY OF THE INVENTION

In accordance with the instant invention, prodrugs of integraseinhibitors are provided. In some embodiments, the prodrug is a dimer ofintegrase inhibitors connected by a linker (e.g., an optionallysubstituted aliphatic or alkyl group). In some embodiments, the prodrugcomprises an integrase inhibitor modified with an amino acid fatty estercomprising an optionally substituted aliphatic or alkyl group (e.g., analiphatic or alkyl comprising about 3 to about 30 carbons). In aparticular embodiment, the aliphatic or alkyl group is the alkyl chainof a fatty acid or a saturated linear aliphatic chain, optionallysubstituted with at least one heteroatom. In a particular embodiment,the integrase inhibitor is selected from the group consisting ofcabotegravir (CAB), raltegravir (RAL), elvitegravir (EVG), dolutegravir(DTG), and bictegravir (BIC). Compositions comprising at least oneprodrug of the instant invention and at least one pharmaceuticallyacceptable carrier are also encompassed by the present invention.

In accordance with another aspect of the instant invention,nanoparticles comprising at least one prodrug of the instant inventionand at least one polymer or surfactant are provided. In a particularembodiment, the prodrug is crystalline. In a particular embodiment, thepolymer or surfactant is an amphiphilic block copolymer such as anamphiphilic block copolymer comprising at least one block ofpoly(oxyethylene) and at least one block of poly(oxypropylene) (e.g.,poloxamer 407). The nanoparticle may comprise a polymer or surfactantlinked to at least one targeting ligand. An individual nanoparticle maycomprise targeted and non-targeted surfactants. In a particularembodiment, the nanoparticles have a diameter of about 100 nm to 1 μm.Compositions comprising at least one nanoparticle of the instantinvention and at least one pharmaceutically acceptable carrier are alsoencompassed by the present invention.

In accordance with another aspect of the instant invention, methods fortreating, inhibiting, and/or preventing a disease or disorder in asubject in need thereof are provided. The methods comprise administeringto the subject at least one prodrug or nanoparticle of the instantinvention, optionally within a composition comprising a pharmaceuticallyacceptable carrier. In a particular embodiment, the disease or disorderis a viral infection (e.g., a retroviral infection). In a particularembodiment, the method further comprises administering at least onefurther therapeutic agent or therapy for the disease or disorder, e.g.,at least one additional anti-HIV compound.

BRIEF DESCRIPTIONS OF THE DRAWING

FIG. 1 provides a schematic of the synthesis of certain prodrugs of theinstant invention.

FIG. 2A provides a graph of HIV-1 reverse transcriptase (RT) activity inhuman monocyte derived macrophages (MDM) treated with the indicatedconcentration of drug and challenged with HIV-1_(ADA). FIG. 2B providesa graph of the cell viability of MDM after nanoparticle (NM3DTG orNM4DTG) treatment at the indicated concentrations. Results werenormalized to untreated control cells.

FIGS. 3A and 3B provide graphs of the drug uptake by MDM over a 24 hourperiod with equal drug concentrations at 25 μM (FIG. 3A) or 50 μM (FIG.3B).

FIGS. 4A and 4B provide graphs of the drug retention by MDM over a 30day period with equal drug concentrations at 25 μM (FIG. 4A) or 50 μM(FIG. 4B).

FIGS. 5A and 5B provide graphs of HIV-1 reverse transcriptase activityat the indicated timepoints in MDM treated with the indicatednanoparticles and challenged with HIV-1_(ADA). MDM were pretreated for 8hours with equal drug concentrations of 1 μM (FIG. 5A) or 10 μM (FIG.5B) NM3DTG or NM4DTG. At the indicated times, cells were challenged withHIV-1_(ADA) and media was collected after an additional 10 days andassayed for HIV-1 RT activity.

FIG. 6A provides a graph of weight of the mice during thepharmacokinetic studies. FIG. 6B provides a graph of plasma DTG levelsafter a single intramuscular (IM) dose of NDTG, NM3DTG, or NM4DTG inBalb/cJ mice. Administered dose was 45 mg DTG equivalents (eq)/kg. Topbold dashed line indicates plasma DTG 4×PA-IC₉₀ of 256 ng/ml and thebottom stippled line shows the plasma DTG PA-IC₉₀ of 64 ng/ml. n=3-5animals/group.

DETAILED DESCRIPTION OF THE INVENTION

Maximal restriction of viral load in tissue infectious sites canfacilitate viral eradication strategies. This can be achieved bygeneration of potent lipophilic and hydrophobic antiretroviral prodrugnanocrystals stabilized by surfactants. Hydrophobicities, drughydrolysis rates, and antiretroviral potencies must be balanced foroptimal therapeutic effect. Herein, dimer prodrugs and amino acid fattyester prodrugs are shown to optimize therapeutic efficacy, particularlywith regard to long acting slow effective release antiretroviral therapy(LASER ART). LASER ART refers to a long acting antiretroviral druggenerated from a nanocrystal prodrug. Herein, it is shown that dimerprodrugs and amino acid fatty ester prodrugs unexpectedly serve toenhance DTG lipophilicity and hydrophobicity, improve drug potency, andslow prodrug hydrolysis, thereby extensively extending the half-life ofthe parent drug. The novel prodrugs enhance drug encapsulation withappropriate excipients and stabilizers, such as poloxamer 407 (P407).The nanoformulations provide sustained drug release and site specificantiretroviral drug delivery. The prodrugs comprise native drugconjugated to hydrophobic moieties via hydrolyzable covalent bonds. Thenanoformulations of the prodrugs of the instant invention were readilytaken up by human monocyte-derived macrophages (MDM) with sustained drugretention for 30 days in vitro; whereas parent drug nanoformulationshowed rapid HIV-1 breakthrough in MDM. Notably, MDM treated withnanoformulations of the prodrugs of the instant invention exhibitedsustained antiretroviral activities following HIV-1 challenge for up to30 days after single drug treatment. Further, a single intramuscular(IM) injection of nanoformulations of the prodrugs of the instantinvention at 45 mg DTG equivalents/kg into mice demonstrated a zeroorder controlled release kinetics of active DTG and provided drug levelsat or above 4 times the PA-IC₉₀ for greater than several months. Thenanoformulations presented herein improves upon current combination ARTregimens that require multiple daily administrations by reducing pillburden, lowering the risk of viral rebound, limiting toxicities, and/orallowing for drug penetration into viral reservoirs. Importantly, thenanoformulations also facilitate a dosing interval of once every threeto six months (or even less frequently) to maximize the effectiveness ofpre-exposure prophylaxis or treatment regimens.

Long acting slow effective release ART (LASER ART) formulations canextend dosing intervals, reduce systemic toxicity, and improvepharmacokinetic (PK) and pharmacodynamic (PD) profiles (Sillman, et al.,Nat. Commun. (2018) 9:443; Zhou, et al., Biomaterials (2018) 151:53-65;McMillan, et al., Antimicrob. Agents Chemother. (2018) 62:e01316-17).Herein, novel integrase inhibitor prodrugs, long-acting slow effectiverelease formulations thereof, and methods of synthesis and use thereofare provided. Integrase inhibitors (integrase strand transfer inhibitors(INSTIs)) are a class of antiretroviral drug designed to block theaction of integrase (e.g., HIV integrase), a viral enzyme that insertsthe viral genome into the DNA of the host cell. Examples of integraseinhibitors include, without limitation, cabotegravir (CAB, GSK1265744),raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG, GSK1349572),bictegravir (BIC, GS-9883), BI 224436 (Boehringer Ingelheim, Ingelheim,Germany), and MK-2048 (Merck, Kenilworth, N.J.). The prodrugs of theinstant invention and their slow effective release formulations exhibitenhanced potency and efficacy, increased cellular and tissue penetrationand extended half-lives compared to parent integrase inhibitor. Theprodrugs and their formulations of the instant invention and theircombinations can be used in the management of viral (e.g., retroviral)infections.

Treatments of viral infections, particularly HIV infections, which arecurrently available, include inhibitors of viral entry, nucleosidereverse transcriptase, nucleotide reverse transcriptase, integrase, andprotease. Resistance is linked to a shortened drug half-life, the virallife cycle, and rapid mutations resulting in a high genetic variability.Combination therapies, e.g., antiretroviral therapies (ART), which areconsidered “cocktail” therapy, have gained substantial attention.Benefits include decreased viral resistance, limited toxicities,improved adherence to therapeutic regimens and sustained antiretroviralefficacy. Combination therapies minimize potential drug resistance bysuppressing viral (e.g., HIV) replication, thereby reducing spontaneousresistant mutants. Treatment failure is attributed, in part, to theshort drug half-lives. Furthermore, failure can also be attributed, inpart, to limited drug access to tissue and cellular viral reservoirs,thereby precluding viral eradication efforts. To these ends, thedevelopment of cell and tissue targeted nanoformulated prodrug(nanoparticle) platforms are of considerable interest in the managementof viral (e.g., HIV) infections. Pre-exposure prophylaxis (PrEP) isanother strategy used in the management of viral (e.g., HIV)transmission. For example, TRUVADA® (tenofovir/emtricitabine) has beenapproved for pre-exposure prophylaxis against HIV infection.Additionally, the combination of lamivudine and zidovudine (COMBIVIR®)has been used as pre-exposure prophylaxis and post-exposure prophylaxis.

The prodrugs and nanoformulated prodrugs (nanoparticles) provided hereinunexpectedly extend the drug half-life, increase hydrophobicity andlipophilicity, and improve antiretroviral efficacy. This will benefitpeople who have to receive daily high doses or even several doses a day,since lower dosage with less dosing frequency would not only decreasethe side effects, but also be convenient to the patients. The prodrugsand nanoformulated prodrugs (nanoparticles) provided herein may also beused as a post-exposure treatment and/or pre-exposure prophylaxis (e.g.,for people who are at high risk of contracting HIV-1). In other words,the prodrugs and nanoparticles of the instant invention and theircombination may be used to prevent a viral infection (e.g., HIVinfection) and/or treat or inhibit an acute or long term viral infection(e.g., HIV infection). While the prodrugs and nanoparticles of theinstant invention are generally described as anti-HIV agents, theprodrugs and nanoformulations of the instant invention are alsoeffective against other viral infections including, without limitation:retroviruses (e.g., lentiviruses), hepatitis B virus (HBV), hepatitis Cvirus (HCV), and human T-cell leukemia viruses (HTLV), particularlyretroviruses.

The present invention describes novel, potent, broad spectrum prodrugswith improved biological activity over parent drugs. Methods for theencapsulation of the prodrugs into long acting slow effectiveformulations for efficient intracellular and tissue delivery andextended drug half-lives are also provided. The long acting sloweffective release (LASER) compositions described herein exhibit enhancedpotency and may be used as effective therapeutic or preventativeinterventions against viral infections (e.g., retroviral infections).

Prodrugs of the instant invention allow for the efficient intracellulardelivery of integrase inhibitors. Herein, prodrugs are provided whichare derivatives of integrase inhibitors. In certain embodiments, achemical moiety of the integrase inhibitor, particularly an oxygencontaining moiety such as a hydroxyl group, has been replaced with anester moiety (e.g., an ester moiety comprising a hydrophobic andlipophilic cleavable moiety). Prodrugs of the instant invention include,but are not limited to: fatty diester and monoester prodrugs, fattyester integrase inhibitor dimer prodrugs, and amino acid fatty esters.

As described herein, the prodrugs can improve drug potency, accelerateintracellular and tissue penetrance, protein binding, andbioavailability. The hydrophobic nature of the synthesized prodrugsfacilitates encapsulation into long acting slow release drugnanocrystals with improved biopharmaceutical features. Thenanoformulations of the instant invention may be composed of prodrugparticles dispersed in sterile aqueous suspensions and stabilized bypolymeric excipients, lipids, and/or surfactants or polymers. Withoutbeing bound by theory, the mechanism of drug release involvesdissolution of the prodrug from the nanoparticle followed by efficientcleavage to generate bioactive agents, e.g., an integrase inhibitorand/or a broad-spectrum antiviral fatty alcohol.

The benefits of the system described herein include, without limitation,improved drug potency, bioavailability and extended half-life forpatient convenience. Indeed, the nanoformulations described in thisinvention displayed significant increase in drug uptake bymonocyte-derived macrophages (MDM). Also, the modified drug andnanoparticles exhibited enhanced potency through increased and extendedinhibition of viral replication. Therefore, the nanoformulations of theinstant invention allow for enhancement of antiviral potency andaccelerated drug delivery to anatomical reservoirs of infection.

In accordance with the instant invention, prodrugs of integraseinhibitors are provided. In a particular embodiment, the integraseinhibitor is selected from the group consisting of cabotegravir (CAB),raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir(BIC), BI 224436, and MK-2048. In a particular embodiment, the integraseinhibitor is selected from the group consisting of cabotegravir (CAB),raltegravir (RAL), elvitegravir (EVG), dolutegravir (DTG), andbictegravir (BIC). In a particular embodiment, the integrase inhibitoris dolutegravir (DTG). In a particular embodiment, the integraseinhibitor is cabotegravir (CAB). Examples of the chemical structures ofthese integrase inhibitors are:

In some embodiments, the prodrug of the present invention is a dimer oftwo integrase inhibitors that are connected by a linker. The integraseinhibitors in the dimer prodrug may be the same integrase inhibitor orthey may be different integrase inhibitors. In a particular embodiment,the prodrug comprises an integrase inhibitor wherein a chemical moiety,particularly an oxygen containing moiety such as a hydroxyl group, isreplaced with an ester comprising the linker. In a particularembodiment, the linker is an optionally substituted aliphatic or alkylgroup. The aliphatic or alkyl group may be unsaturated or saturated, andmay be substituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, the alkyl or aliphatic group is hydrophobic. In aparticular embodiment, the linker is an optionally substitutedhydrocarbon chain, particularly saturated. In a particular embodiment,the linker a hydrocarbon chain. In a particular embodiment, the linkeris a saturated linear aliphatic chain. In a particular embodiment, thealkyl or aliphatic group comprises about 1 to about 30 carbons (e.g., inthe main chain of the alkyl or aliphatic group), which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, the linker is 1 to about 30 carbon atoms inlength, 1 to about 28 carbons in length, 1 to about 26 carbons inlength, 1 to about 24 carbons in length, 1 to about 22 carbons inlength, 1 to about 20 carbons in length, 1 to about 18 carbons inlength, 1 to about 16 carbons in length, 1 to about 10 carbons inlength, 10 to about 22 carbons in length, 10 to about 20 carbons inlength, 12 to about 20 carbons in length, 14 to about 18 carbons inlength, or about 16 carbons in length. Numbering here excludes thecarbon in the C═O of the ester.

In a particular embodiment, the prodrug of the instant invention isselected from the following group or a pharmaceutically acceptable saltor stereoisomer thereof:

wherein n is from 1 to about 24, 1 to about 22, 1 to about 20, 1 toabout 18, 1 to about 16, 1 to about 14, 1 to about 12, 1 to about 10, 1to about 4, 4 to about 16, 4 to about 14, 6 to about 14, 8 to about 12,or about 10. In a particular embodiment, the linker may be substitutedwith at least one heteroatom (e.g., O, N, or S).

In a particular embodiment, the prodrug of the instant invention is:

(M3DTG) or a pharmaceutically acceptable salt or stereoisomer thereof.In a particular embodiment, the one or both of the DTG is replaced withCAB. In a particular embodiment, the prodrug is M3CAB or M4CAB, or apharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, the prodrug of the present invention is an aminoacid fatty ester. In a particular embodiment, the prodrug comprises anintegrase inhibitor wherein a chemical moiety, particularly an oxygencontaining moiety such as a hydroxyl group, is replaced with an aminoacid fatty ester. The amino acid fatty ester may contain one or moreamino acids, residues or side chains. In a particular embodiment, theamino fatty ester comprises 1 to 10 amino acids, particularly 1 to 7amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino acids,1 to 2 amino acids, or 1 amino acid. In a particular embodiment, theamino fatty ester comprises only one amino acid, residue, or side chain.In a particular embodiment, the amino acid forms an amide bond with theC═O of the ester. In a particular embodiment, the prodrug comprises anintegrase inhibitor wherein a hydroxyl group is replaced with the O ofthe amino acid carboxyl (—COOH) group. Any amino acid may be used. Theamino acids of the amino acid fatty ester may be the same or different.In a particular embodiment, the amino acid is not charged (e.g., notaspartic acid, glutamic acid, arginine, lysine, or histidine). In aparticular embodiment, the amino acid is hydrophobic. In a particularembodiment, the amino acid is selected from the group consisting ofglycine, alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, and tryptophan. In a particular embodiment, the amino acidis selected from the group consisting of alanine, valine, phenylalanine,proline, tyrosine, and lysine. In a particular embodiment, the aminoacid is proline. In a particular embodiment, the amino acid fatty estercomprises a hydrophobic and lipophilic cleavable moiety (e.g.,therapeutic fatty alcohols). The hydrophobic and lipophilic cleavablemoiety (e.g., therapeutic fatty alcohols) can exhibit antiviral activityagainst enveloped viruses (Katz, et al., Ann. NY Acad. Sci. (1994)724:472-88). Notably, synergistic interactions between therapeutic fattyalcohols and nucleoside analogs can substantially enhance antiviralpotency of the nucleosides (Marcelletti, et al., Antiviral Res. (2002)56:153-66).

The hydrophobic nature of the synthesized prodrugs facilitatesencapsulation into long acting slow release drug nanocrystals withimproved biopharmaceutical features. The nanoformulations of the instantinvention may be composed of prodrug particles dispersed in sterileaqueous suspensions and stabilized by polymeric excipients, lipids,and/or surfactants or polymers. Without being bound by theory, themechanism of drug release involves dissolution of the prodrug from thenanoparticle followed by efficient cleavage to generate two bioactiveagents, i.e., the integrase inhibitor and the broad-spectrum antiviralfatty alcohols.

In a particular embodiment, the prodrug of the instant invention isselected from the following group or a pharmaceutically acceptable saltor stereoisomer thereof:

wherein R is an optionally substituted aliphatic or alkyl, and whereinAA is one or more amino acids, residues, or side chains. In a particularembodiment, the 0 attached to AA in the above formulas is the 0 of thehydroxyl in the amino acid carboxyl (—COOH) group. In a particularembodiment, the amino acid forms an amide bond. In a particularembodiment, the amino fatty ester comprises 1 to 10 amino acids,particularly 1 to 7 amino acids, 1 to 5 amino acids, 1 to 4 amino acids,1 to 3 amino acids, 1 to 2 amino acids, or 1 amino acid. In a particularembodiment, the amino fatty ester comprises only one amino acid. Anyamino acid may be used. The amino acids of the amino acid fatty estermay be the same or different. In a particular embodiment, the amino acidis not charged (e.g., not aspartic acid, glutamic acid, arginine,lysine, or histidine). In a particular embodiment, the amino acid ishydrophobic. In a particular embodiment, the amino acid is selected fromthe group consisting of alanine, valine, phenylalanine, proline,tyrosine, and lysine.

In a particular embodiment, R is the side chain of a fatty acid. Thealiphatic or alkyl group may be unsaturated or saturated, and may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R may contain an aromatic moiety that may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R has between 1 and 24 carbons. In a particularembodiment, R has between 10 and 24 carbons.

In a particular embodiment, the alkyl or aliphatic group is hydrophobic.In a particular embodiment, R is an optionally substituted hydrocarbonchain, particularly saturated. In a particular embodiment, R is asaturated linear aliphatic chain. In a particular embodiment, the alkylor aliphatic group comprises about 1 to about 30 carbons, about 1 toabout 24 carbons, or about 10 to about 24 carbons (e.g., in the mainchain of the alkyl or aliphatic group), which may be substituted with atleast one heteroatom (e.g., O, N, or S). In a particular embodiment, Ris a C1-C29 unsaturated or saturated alkyl or aliphatic group, which maybe substituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C1-C21 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C9-C29unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C9-C21 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C7-C23unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C9-C21 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C11-C19unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C13-C19 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C13-C17unsaturated or saturated alkyl or aliphatic group, which may besubstituted with at least one heteroatom (e.g., O, N, or S). In aparticular embodiment, R is a C17 unsaturated or saturated alkyl oraliphatic group, which may be substituted with at least one heteroatom(e.g., O, N, or S). In a particular embodiment, R is a C15 unsaturatedor saturated alkyl or aliphatic group, which may be substituted with atleast one heteroatom (e.g., O, N, or S).

In a particular embodiment, R is the alkyl chain of a fatty acid(saturated or unsaturated), particularly a C4-C30 fatty acid, C6-C28fatty acid, C8-C26 fatty acid a C10-C24 fatty acid, a C12-C22 fattyacid, a C14-C22 fatty acid, a C14-C20 fatty acid, a C14-C18 fatty acid,a C16-C18 fatty acid, a C18 fatty acid, or a C16 fatty acid (numberinghere is inclusive of the carbon in the C═O of the ester).

In a particular embodiment, R is a saturated linear aliphatic chain or ahydrocarbon chain of at least 9 carbons (e.g., 9 to 21 carbons in lengthin the chain, 9 to 19 carbons in length in the chain, 11 to 17 carbonsin length in the chain, 13 to 21 carbons in length in the chain, 13 to19 carbons in length in the chain, 15 to 17 carbons in length in thechain, or 15 or 17 carbons in length in the chain). In a particularembodiment, R is a saturated linear aliphatic chain or a hydrocarbonchain of 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 carbons in length,particularly 12, 13, 14, 15, 16, 17, 18, or 19 carbons in length, 15,16, or 17 carbons in length, or 15 carbons in length. In a particularembodiment, R is a saturated linear aliphatic chain or a hydrocarbonchain of 15 carbons in length.

In a particular embodiment, the prodrug of the instant invention is:

or a pharmaceutically acceptable salt or stereoisomer thereof. In aparticular embodiment, the DTG is replaced with CAB. In a particularembodiment, the prodrug is M5CAB or M6CAB, or a pharmaceuticallyacceptable salt or stereoisomer thereof.

The instant invention also encompasses nanoparticles (sometimes referredto herein as nanoformulations) comprising the prodrug of the instantinvention. The nanoparticles may be used for the delivery of thecompounds to a cell or host (e.g., in vitro or in vivo). In a particularembodiment, the nanoparticle is used for the delivery of antiretroviraltherapy to a subject. The nanoparticles of the instant inventioncomprise at least one prodrug and at least one surfactant or polymer. Ina particular embodiment, the nanoparticles comprise aspectroscopic-defined surfactant/polymer:drug ratio that maintainsoptimal targeting of the drug nanoparticle to maintain a macrophagedepot. These components of the nanoparticle, along with other optionalcomponents, are described hereinbelow.

Methods of synthesizing the nanoparticles of the instant invention areknown in the art. In a particular embodiment, the methods generatenanoparticles comprising a prodrug (e.g., crystalline or amorphous)coated (either partially or completely) with a polymer and/orsurfactant. Examples of synthesis methods include, without limitation,milling (e.g., wet milling), homogenization (e.g., high pressurehomogenization), particle replication in nonwetting template (PRINT)technology, and/or sonication techniques. For example, U.S. PatentApplication Publication No. 2013/0236553, incorporated by referenceherein, provides methods suitable for synthesizing nanoparticles of theinstant invention. In a particular embodiment, the polymers orsurfactants are firstly chemically modified with targeting ligands andthen used directly or mixed with non-targeted polymers or surfactants incertain molar ratios to coat on the surface of prodrug suspensions—e.g.,by using a nanoparticle synthesis process (e.g., a crystallinenanoparticle synthesis process) such as milling (e.g., wet milling),homogenization (e.g., high pressure homogenization), particlereplication in nonwetting template (PRINT) technology, and/or sonicationtechniques, thereby preparing targeted nanoformulations. Thenanoparticles may be used with or without further purification, althoughthe avoidance of further purification is desirable for quickerproduction of the nanoparticles. In a particular embodiment, thenanoparticles are synthesized using milling and/or homogenization.Targeted nanoparticles (e.g., using ligands (optionally with highmolecular weight)) may be developed through either physically orchemically coating and/or binding on the surface of polymers orsurfactants and/or drug nanosuspensions.

In a particular embodiment, the nanoparticles of the instant inventionare synthesized by adding the prodrug (e.g., crystals) to a polymer orsurfactant solution and then generating the nanoparticles (e.g., by wetmilling or high pressure homogenization). The prodrug and polymer orsurfactant solution may be agitated prior to the wet milling or highpressure homogenization.

The nanoparticles of the instant invention may be used to deliver atleast one prodrug of the instant invention to a cell or a subject(including non-human animals). In a particular embodiment, thenanoparticle comprises more than one dimer prodrug (i.e., at least twounique dimer prodrugs). In a particular embodiment, the nanoparticlecomprises more than one amino acid fatty ester prodrug (i.e., at leasttwo unique amino acid fatty ester prodrugs). In a particular embodiment,the nanoparticle comprises at least one amino acid fatty ester prodrugand at least one dimer prodrug. The nanoparticles of the instantinvention may further comprise at least one other agent or compound,particularly a bioactive agent, particularly a therapeutic agent (e.g.,antiviral compound) or diagnostic agent, particularly at least oneantiviral or antiretroviral. In a particular embodiment, thenanoparticles of the instant invention comprise at least two therapeuticagents, particularly wherein at least one is a prodrug of the instantinvention. For example, the nanoparticle may comprise an integraseinhibitor prodrug of the instant invention and at least one othertherapeutic agent (e.g., an anti-HIV agent).

In a particular embodiment, the nanoparticles of the instant inventionare a submicron colloidal dispersion of nanosized prodrug crystalsstabilized by polymers or surfactants (e.g., surfactant-coated drugcrystals; a nanoformulation). In a particular embodiment, the prodrugmay be crystalline (solids having the characteristics of crystals),amorphous, or are solid-state nanoparticles of the prodrug that isformed as crystal that combines the drug and polymer or surfactant. In aparticular embodiment, the prodrug is crystalline. As used herein, theterm “crystalline” refers to an ordered state (i.e., non-amorphous)and/or a substance exhibiting long-range order in three dimensions. In aparticular embodiment, the majority (e.g., at least 50%, 60%, 70%, 80%,90%, 95% or more) of the prodrug and, optionally, the hydrophobicportion of the surfactant or polymer are crystalline.

In a particular embodiment, the nanoparticle of the instant invention isup to about 2 or 3 μm in diameter (e.g., z-average diameter) or itslongest dimension, particularly up to about 1 μm (e.g., about 100 nm toabout 1 μm). For example, the diameter or longest dimension of thenanoparticle may be about 50 to about 800 nm. In a particularembodiment, the diameter or longest dimension of the nanoparticle isabout 50 to about 750 nm, about 50 to about 600 nm, about 50 to about500 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm,about 200 nm to about 400 nm, about 250 nm to about 350 nm, or about 250nm to about 400 nm. The nanoparticles may be, for example, rod shaped,elongated rods, irregular, or round shaped. The nanoparticles of theinstant invention may be neutral or charged. The nanoparticles may becharged positively or negatively.

As stated hereinabove, the nanoparticles of the instant inventioncomprise at least one polymer or surfactant. A “surfactant” refers to asurface-active agent, including substances commonly referred to aswetting agents, detergents, dispersing agents, or emulsifying agents.Surfactants are usually organic compounds that are amphiphilic.

Examples of polymers or surfactants include, without limitation,synthetic or natural phospholipids, PEGylated lipids (e.g., PEGylatedphospholipid), lipid derivatives, polysorbates, amphiphilic copolymers,amphiphilic block copolymers, poly(ethyleneglycol)-co-poly(lactide-co-glycolide) (PEG-PLGA), their derivatives,ligand-conjugated derivatives and combinations thereof. Other polymersor surfactants and their combinations that can form stablenanosuspensions and/or can chemically/physically bind to the targetingligands of HIV infectable/infected CD4+ T cells, macrophages anddendritic cells can be used in the instant invention. Further examplesof polymers or surfactants include, without limitation: 1) nonionicsurfactants (e.g., pegylated and/or polysaccharide-conjugated polyestersand other hydrophobic polymeric blocks such aspoly(lactide-co-glycolide) (PLGA), polylactic acid (PLA),polycaprolactone (PCL), other polyesters, poly(propylene oxide),poly(1,2-butylene oxide), poly(n-butylene oxide),poly(tetrahydrofurane), and poly(styrene); glyceryl esters,polyoxyethylene fatty alcohol ethers, polyoxyethylene sorbitan fattyacid esters, polyoxyethylene fatty acid esters, sorbitan esters,glycerol monostearate, polyethylene glycols, polypropyleneglycols, cetylalcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyetheralcohols, polyoxyethylene-polyoxypropylene copolymers, poloxamines,cellulose, methylcellulose, hydroxylmethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose, polysaccharides,starch and their derivatives, hydroxyethylstarch, polyvinyl alcohol(PVA), polyvinylpyrrolidone, and their combination thereof); and 2)ionic surfactants (e.g., phospholipids, amphiphilic lipids,1,2-dialkylglycero-3-alkylphophocholines, 1,2-distearoyl-sn-glecro-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol) (DSPE-PEG), dimethylaminoethanecarbamoyl cheolesterol (DC-Chol),N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), alkylpyridinium halides, quaternary ammonium compounds,lauryldimethylbenzylammonium, acyl carnitine hydrochlorides,dimethyldioctadecylammonium (DDAB), n-octylamines, oleylamines,benzalkonium, cetyltrimethylammonium, chitosan, chitosan salts,poly(ethylenimine) (PEI), poly(N-isopropyl acrylamide (PNIPAM), andpoly(allylamine) (PAH), poly (dimethyldiallylammonium chloride) (PDDA),alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassiumlaurate, triethanolamine stearate, sodium lauryl sulfate, sodiumdodecylsulfate, alkyl polyoxyethylene sulfates, alginic acid, alginicacid salts, hyaluronic acid, hyaluronic acid salts, gelatins, dioctylsodium sulfosuccinate, sodium carboxymethylcellulose, cellulose sulfate,dextran sulfate and carboxymethylcellulose, chondroitin sulfate,heparin, synthetic poly(acrylic acid) (PAA), poly (methacrylic acid)(PMA), poly(vinyl sulfate) (PVS), poly(styrene sulfonate) (PSS), bileacids and their salts, cholic acid, deoxycholic acid, glycocholic acid,taurocholic acid, glycodeoxycholic acid, derivatives thereof, andcombinations thereof.

The polymer or surfactant of the instant invention may be charged orneutral. In a particular embodiment, the polymer or surfactant isneutral or negatively charged (e.g., poloxamers, polysorbates,phospholipids, and their derivatives).

In a particular embodiment, the polymer or surfactant is an amphiphilicblock copolymer or lipid derivative. In a particular embodiment, atleast one polymer or surfactant of the nanoparticle is an amphiphilicblock copolymer, particularly a copolymer comprising at least one blockof poly(oxyethylene) and at least one block of poly(oxypropylene). In aparticular embodiment, the polymer or surfactant is a triblockamphiphilic block copolymer. In a particular embodiment, the polymer orsurfactant is a triblock amphiphilic block copolymer comprising acentral hydrophobic block of polypropylene glycol flanked by twohydrophilic blocks of polyethylene glycol. In a particular embodiment,the surfactant is poloxamer 407.

In a particular embodiment, the amphiphilic block copolymer is acopolymer comprising at least one block of poly(oxyethylene) and atleast one block of poly(oxypropylene). In a particular embodiment, theamphiphilic block copolymer is a poloxamer. Examples of poloxamersinclude, without limitation, Pluronic® L31, L35, F38, L42, L43, L44,L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88,L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5,10R8, 12R3, 17R1, 17R2, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8,31R1, 31R2, and 31R4. In a particular embodiment, the poloxamer ispoloxamer 407 (Pluronic® F127).

In a particular embodiment of the invention, the polymer or surfactantis present in the nanoparticle and/or solution to synthesize thenanoparticle (as described herein) at a concentration ranging from about0.0001% to about 10% or 15% by weight. In a particular embodiment, theconcentration of the polymer or surfactant ranges from about 0.01% toabout 15%, about 0.01% to about 10%, about 0.1% to about 10%, or about0.1% to about 6% by weight. In a particular embodiment, the nanoparticlecomprises at least about 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% orhigher therapeutic agent (prodrug) by weight. In a particularembodiment, the nanoparticles comprise a defined drug:polymer/surfactantratio. In a particular embodiment, the drug:polymer/surfactant ratio(e.g., by weight) is from about 1:1 to about 1000:1, about 1:1 to about10:1, about 10:6 to about 1000:6, about 20:6 to about 500:6, about 50:6to about 200:6, or about 100:6.

As stated hereinabove, the polymer or surfactant of the instantinvention may be linked to a targeting ligand. The targeting of thenanoparticles (e.g., to macrophage) can provide for superior targeting,decreased excretion rates, decreased toxicity, and prolonged half-lifecompared to free drug or non-targeted nanoparticles. A targeting ligandis a compound that specifically binds to a specific type of tissue orcell type (e.g., in a desired target:cell ratio). For example, atargeting ligand may be used for engagement or binding of a target cell(e.g., a macrophage, T cell, dendritic cell, etc.) surface marker orreceptor which may facilitate its uptake into the cell (e.g., within aprotected subcellular organelle that is free from metabolicdegradation). In a particular embodiment, the targeting ligand is aligand for a cell surface marker/receptor. The targeting ligand may bean antibody or antigen-binding fragment thereof immunologically specificfor a cell surface marker (e.g., protein or carbohydrate) preferentiallyor exclusively expressed on the targeted tissue or cell type. Thetargeting ligand may be linked directly to the polymer or surfactant orvia a linker. Generally, the linker is a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches the ligand tothe polymer or surfactant. The linker can be linked to any syntheticallyfeasible position of the ligand and the polymer or surfactant. Exemplarylinkers may comprise at least one optionally substituted; saturated orunsaturated; linear, branched or cyclic aliphatic group, an alkyl group,or an optionally substituted aryl group. The linker may be a lower alkylor aliphatic. The linker may also be a polypeptide (e.g., from about 1to about 10 amino acids, particularly about 1 to about 5). In aparticular embodiment, the targeting moiety is linked to either or bothends of the polymer or surfactant. The linker may be non-degradable andmay be a covalent bond or any other chemical structure which cannot besubstantially cleaved or cleaved at all under physiological environmentsor conditions.

The nanoparticles/nanoformulations of the instant invention may comprisetargeted and/or non-targeted polymers or surfactants. In a particularembodiment, the molar ratio of targeted and non-targeted polymers orsurfactants in the nanoparticles/nanoformulations of the instantinvention is from about 0.001 to 100%, about 1% to about 99%, about 5%to about 95%, about 10% to about 90%, about 25% to about 75%, about 30%to about 60%, or about 40%. In a particular embodiment, the nanoparticlecomprises only targeted polymers or surfactants. In a particularembodiment, the nanoparticles/nanoformulations of the instant inventioncomprise a folate targeted polymer or surfactant and a non-targetedversion of the polymer or surfactant. In a particular embodiment, thenanoparticles/nanoformulations of the instant invention comprisefolate-poloxamer 407 (FA-P407) and/or poloxamer 407.

Examples of targeting ligands include but are not limited to macrophagetargeting ligands, CD4+ T cell targeting ligands, dendritic celltargeting ligands, and tumor targeting ligands. In a particularembodiment, the targeting ligand is a macrophage targeting ligand. Thetargeted nanoformulations of the instant invention may comprise atargeting ligand for directing the nanoparticles to HIV tissue andcellular sanctuaries/reservoirs (e.g., central nervous system, gutassociated lymphoid tissues (GALT), CD4+ T cells, macrophages, dendriticcells, etc.). Macrophage targeting ligands include, without limitation,folate receptor ligands (e.g., folate (folic acid) and folate receptorantibodies and fragments thereof (see, e.g., Sudimack et al. (2000) Adv.Drug Del. Rev., 41:147-162)), mannose receptor ligands (e.g., mannose),formyl peptide receptor (FPR) ligands (e.g., N-formyl-Met-Leu-Phe(fMLF)), and tuftsin (the tetrapeptide Thr-Lys-Pro-Arg). Other targetingligands include, without limitation, hyaluronic acid, gp120 and peptidefragments thereof, and ligands or antibodies specific for CD4, CCR5,CXCR4, CD7, CD111, CD204, CD49a, CD29, CD19, CD20, CD22, CD171, CD33,Leis-Y, WT-1, ROR1, MUC16, MUC1, MUC4, estrogen receptor, transferrinreceptors, EGF receptors (e.g. HER2), folate receptor, VEGF receptor,FGF receptor, androgen receptor, NGR, Integrins, and GD2. In aparticular embodiment, the targeting ligand is folic acid.

As stated hereinabove, the nanoparticles of the instant invention may tocomprise a further therapeutic agent. The instant invention alsoencompasses therapeutic methods wherein the prodrug and/or nanoparticlesof the instant invention are co-administered with another therapeuticagent. In a particular embodiment, the therapeutic agent is hydrophobic,a water insoluble compound, or a poorly water soluble compound,particularly when included in the nanoparticle. For example, thetherapeutic agent may have a solubility of less than about 10 mg/ml,less than 1 mg/ml, more particularly less than about 100 μg/ml, and moreparticularly less than about 25 μg/ml in water or aqueous media in a pHrange of 0-14, preferably between pH 4 and 10, particularly at 20° C.

In a particular embodiment, the therapeutic agent is an antiviral or anantiretroviral. The antiretroviral may be effective against or specificto lentiviruses. Lentiviruses include, without limitation, humanimmunodeficiency virus (HIV) (e.g., HIV-1, HIV-2), bovineimmunodeficiency virus (BIV), feline immunodeficiency virus (FIV),simian immunodeficiency virus (SIV), and equine infectious anemia virus(EIA). In a particular embodiment, the therapeutic agent is an anti-HIVagent. An anti-HIV compound or an anti-HIV agent is a compound whichinhibits HIV (e.g., inhibits HIV replication and/or infection). Examplesof anti-HIV agents include, without limitation:

(I) Nucleoside-analog reverse transcriptase inhibitors (NRTIs). NRTIsrefer to nucleosides and nucleotides and analogues thereof that inhibitthe activity of reverse transcriptase, particularly HIV-1 reversetranscriptase. NRTIs comprise a sugar and base. Examples ofnucleoside-analog reverse transcriptase inhibitors include, withoutlimitation, adefovir dipivoxil, adefovir, lamivudine, telbivudine,entecavir, tenofovir, stavudine, abacavir, didanosine, emtricitabine,zalcitabine, and zidovudine.

(II) Non-nucleoside reverse transcriptase inhibitors (NNRTIs). NNRTIsare allosteric inhibitors which bind reversibly at anonsubstrate-binding site on reverse transcriptase, particularly the HIVreverse transcriptase, thereby altering the shape of the active site orblocking polymerase activity. Examples of NNRTIs include, withoutlimitation, delavirdine (DLV, BHAP, U-90152; Rescriptor®), efavirenz(EFV, DMP-266, SUSTIVA®), nevirapine (NVP, Viramune®), PNU-142721,capravirine (S-1153, AG-1549), emivirine (+)-calanolide A (NSC-675451)and B, etravirine (ETR, TMC-125, Intelence®), rilpivirne (RPV, TMC278,Edurant™) DAPY (TMC120), doravirine (Pifeltro™), BILR-355 BS, PHI-236,and PHI-443 (TMC-278).

(III) Protease inhibitors (PI). Protease inhibitors are inhibitors of aviral protease, particularly the HIV-1 protease. Examples of proteaseinhibitors include, without limitation, darunavir, amprenavir (141W94,AGENERASE®), tipranivir (PNU-140690, APTIVUS®), indinavir (MK-639;CRIXIVAN®), saquinavir (INVIRASE®, FORTOVASE®), fosamprenavir (LEXIVA®),lopinavir (ABT-378), ritonavir (ABT-538, NORVIR®), atazanavir(REYATAZ®), nelfinavir (AG-1343, VIRACEPT®), lasinavir(BMS-234475/CGP-61755), BMS-2322623, GW-640385X (VX-385), AG-001859, andSM-309515.

(IV) Fusion or entry inhibitors. Fusion or entry inhibitors arecompounds, such as peptides, which block HIV entry into a cell (e.g., bybinding to HIV envelope protein and blocking the structural changesnecessary for the virus to fuse with the host cell). Examples of fusioninhibitors include, without limitation, CCR5 receptor antagonists (e.g.,maraviroc (Selzentry®, Celsentri)), enfuvirtide (INN, FUZEON®), T-20(DP-178, FUZEON®) and T-1249.

(V) Integrase inhibitors (in addition to the prodrug of the instantinvention). Integrase inhibitors are a class of antiretroviral drugdesigned to block the action of integrase (e.g., HIV integrase), a viralenzyme that inserts the viral genome into the DNA of the host cell.Examples of integrase inhibitors include, without limitation,raltegravir, elvitegravir, GSK1265744 (cabotegravir), GSK1349572(dolutegravir), GS-9883 (bictegravir), and MK-2048.

Anti-HIV compounds also include maturation inhibitors (e.g., bevirimat).Maturation inhibitors are typically compounds which bind HIV gag anddisrupt its processing during the maturation of the virus. Anti-HIVcompounds also include HIV vaccines such as, without limitation, ALVAC®HIV (vCP1521), AIDSVAX® B/E (gp120), and combinations thereof. Anti-HIVcompounds also include HIV antibodies (e.g., antibodies against gp120 orgp41), particularly broadly neutralizing antibodies.

More than one anti-HIV agent may be used, particularly where the agentshave different mechanisms of action (as outlined above). For example,anti-HIV agents which are not NNRTIs may be combined with the NNRTIprodrugs of the instant invention. In a particular embodiment, theanti-HIV therapy is highly active antiretroviral therapy (HAART).

The instant invention encompasses compositions (e.g., pharmaceutical tocompositions) comprising at least one prodrug and/or nanoparticle of theinstant invention and at least one pharmaceutically acceptable carrier.As stated hereinabove, the nanoparticle may comprise more than onetherapeutic agent. In a particular embodiment, the pharmaceuticalcomposition comprises a first nanoparticle comprising a first prodrugand a second nanoparticle comprising a second prodrug, wherein the firstand second prodrugs are different. In a particular embodiment, the firstprodrug is a prodrug of the instant invention and the second prodrug isa prodrug of a non-nucleoside reverse transcriptase inhibitor (NNRTI),particularly rilpivirine (RPV). The compositions (e.g., pharmaceuticalcompositions) of the instant invention may further comprise othertherapeutic agents (e.g., other anti-HIV compounds (e.g., thosedescribed herein)).

The present invention also encompasses methods for preventing,inhibiting, and/or treating a disease or disorder. The methods compriseadministering a prodrug and/or nanoparticle of the instant invention(optionally in a composition) to a subject in need thereof. In aparticular embodiment, the disease or disorder is a viral (e.g.,retroviral) infection. Examples of viral infections include, withoutlimitation: HIV, Hepatitis B, Hepatitis C, and HTLV. In a particularembodiment, the viral infection is a retroviral or lentiviral infection,particularly an HIV infection (e.g., HIV-1).

The prodrugs and/or nanoparticles of the instant invention (optionallyin a composition) can be administered to an animal, in particular amammal, more particularly a human, in order to treat/inhibit/prevent thedisease or disorder (e.g., a retroviral infection such as an HIVinfection). The pharmaceutical compositions of the instant invention mayalso comprise at least one other therapeutic agent such as an antiviralagent, particularly at least one other anti-HIV compound/agent. Theadditional anti-HIV compound may also be administered in a separatepharmaceutical composition from the prodrugs or compositions of theinstant invention. The pharmaceutical compositions may be administeredat the same time or at different times (e.g., sequentially).

The dosage ranges for the administration of the prodrugs, nanoparticles,and/or compositions of the invention are those large enough to producethe desired effect (e.g., curing, relieving, treating, and/or preventingthe disease or disorder (e.g., HIV infection), the symptoms of it (e.g.,AIDS, ARC), or the predisposition towards it). In a particularembodiment, the pharmaceutical composition of the instant invention isadministered to the subject at an amount from about 5 μg/kg to about 500mg/kg. In a particular embodiment, the pharmaceutical composition of theinstant invention is administered to the subject at an amount greaterthan about 5 μg/kg, greater than about 50 μg/kg, greater than about 0.1mg/kg, greater than about 0.5 mg/kg, greater than about 1 mg/kg, orgreater than about 5 mg/kg. In a particular embodiment, thepharmaceutical composition of the instant invention is administered tothe subject at an amount from about 0.5 mg/kg to about 100 mg/kg, about10 mg/kg to about 100 mg/kg, or about 15 mg/kg to about 50 mg/kg. Thedosage should not be so large as to cause significant adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any counter indications.

The prodrugs and nanoparticles described herein will generally beadministered to a patient as a pharmaceutical composition. The term“patient” as used herein refers to human or animal subjects. Theseprodrugs and nanoparticles may be employed therapeutically, under theguidance of a physician.

The pharmaceutical compositions comprising the prodrugs and/ornanoparticles of the instant invention may be conveniently formulatedfor administration with any pharmaceutically acceptable carrier(s). Forexample, the complexes may be formulated with an acceptable medium suchas water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents, or suitablemixtures thereof, particularly an aqueous solution. The concentration ofthe prodrugs and/or nanoparticles in the chosen medium may be varied andthe medium may be chosen based on the desired route of administration ofthe pharmaceutical composition. Except insofar as any conventional mediaor agent is incompatible with the nanoparticles to be administered, itsuse in the pharmaceutical composition is contemplated.

The dose and dosage regimen of prodrugs and/or nanoparticles accordingto the invention that are suitable for administration to a particularpatient may be determined by a physician considering the patient's age,sex, weight, general medical condition, and the specific condition forwhich the nanoparticles are being administered and the severity thereof.The physician may also take into account the route of administration,the pharmaceutical carrier, and the nanoparticle's biological activity.

Selection of a suitable pharmaceutical composition will also depend uponthe mode of administration chosen. For example, the nanoparticles of theinvention may be administered by direct injection or intravenously. Inthis instance, a pharmaceutical composition comprises the prodrug and/ornanoparticle dispersed in a medium that is compatible with the site ofinjection.

Prodrugs and/or nanoparticles of the instant invention may beadministered by any method. For example, the prodrugs and/ornanoparticles of the instant invention can be administered, withoutlimitation parenterally, subcutaneously, orally, topically, pulmonarily,rectally, vaginally, intravenously, intraperitoneally, intrathecally,intracerbrally, epidurally, intramuscularly, intradermally, orintracarotidly. In a particular embodiment, the prodrug and/ornanoparticle is parenterally. In a particular embodiment, the prodrugand/or nanoparticle is administered orally, intramuscularly,subcutaneously, or to the bloodstream (e.g., intravenously). In aparticular embodiment, the prodrug and/or nanoparticle is administeredintramuscularly or subcutaneously. Pharmaceutical compositions forinjection are known in the art. If injection is selected as a method foradministering the prodrug and/or nanoparticle, steps must be taken toensure that sufficient amounts of the molecules or cells reach theirtarget cells to exert a biological effect. Dosage forms for oraladministration include, without limitation, tablets (e.g., coated anduncoated, chewable), gelatin capsules (e.g., soft or hard), lozenges,troches, solutions, emulsions, suspensions, syrups, elixirs,powders/granules (e.g., reconstitutable or dispersible) gums, andeffervescent tablets. Dosage forms for parenteral administrationinclude, without limitation, solutions, emulsions, suspensions,dispersions and powders/granules for reconstitution. Dosage forms fortopical administration include, without limitation, creams, gels,ointments, salves, patches and transdermal delivery systems.

Pharmaceutical compositions containing a prodrug and/or nanoparticle ofthe present invention as the active ingredient in intimate admixturewith a pharmaceutically acceptable carrier can be prepared according toconventional pharmaceutical compounding techniques. The carrier may takea wide variety of forms depending on the form of pharmaceuticalcomposition desired for administration, e.g., intravenous, oral, directinjection, intracranial, and intravitreal.

A pharmaceutical composition of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical composition appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art. In aparticular embodiment, the prodrugs and/or nanoparticles of the instantinvention, due to their long-acting therapeutic effect, may beadministered once every 1 to 12 months or even less frequently. Forexample, the nanoformulations of the instant invention may beadministered once every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15,18, 21, 24, or more months. In a particular embodiment, the prodrugsand/or nanoparticles of the instant invention are administered less thanonce every two months. In a particular embodiment, the prodrugs and/ornanoformulations of the prodrugs are administered once every month, onceevery two months, particularly once every three months, once every fourmonths, once every five months, once every six months, once every sevenmonths, once every eight months, once every nine months, once every tenmonths, once every eleven months, once every twelve months, or lessfrequently.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of nanoparticles may be determined by evaluatingthe toxicity of the molecules or cells in animal models. Variousconcentrations of nanoparticles in pharmaceutical composition may beadministered to mice, and the minimal and maximal dosages may bedetermined based on the beneficial results and side effects observed asa result of the treatment. Appropriate dosage unit may also bedetermined by assessing the efficacy of the nanoparticle treatment incombination with other standard drugs. The dosage units of nanoparticlemay be determined individually or in combination with each treatmentaccording to the effect detected.

The pharmaceutical composition comprising the nanoparticles may beadministered at appropriate intervals until the pathological symptomsare reduced or alleviated, after which the dosage may be reduced to amaintenance level. The appropriate interval in a particular case wouldnormally depend on the condition of the patient.

The instant invention encompasses methods of treating a disease/disordercomprising administering to a subject in need thereof a pharmaceuticalcomposition comprising a prodrug and/or nanoparticle of the instantinvention and, preferably, at least one pharmaceutically acceptablecarrier. The instant invention also encompasses methods wherein thesubject is treated via ex vivo therapy. In particular, the methodcomprises removing cells from the subject, exposing/contacting the cellsin vitro to the nanoparticles of the instant invention, and returningthe cells to the subject. In a particular embodiment, the cells comprisemacrophage. Other methods of treating the disease or disorder may becombined with the methods of the instant invention may beco-administered with the pharmaceutical compositions of the instantinvention.

The instant also encompasses delivering the nanoparticle of the instantinvention to a cell in vitro (e.g., in culture). The nanoparticle may bedelivered to the cell in at least one carrier.

Definitions

The following definitions are provided to facilitate an understanding ofthe present invention.

The singular forms “a,” “an,” and “the” include plural referents unlessthe context clearly dictates otherwise.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier,buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial, bulkingsubstance (e.g., lactose, mannitol), excipient, auxiliary agent orvehicle with which an active agent of the present invention isadministered. Pharmaceutically acceptable carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin. Water or aqueous saline solutions andaqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. Suitable pharmaceuticalcarriers are described in “Remington's Pharmaceutical Sciences” by E. W.Martin (Mack Publishing Co., Easton, Pa.); Gennaro, A. R., Remington:The Science and Practice of Pharmacy, (Lippincott, Williams andWilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, MarcelDecker, New York, N.Y.; and Kibbe, et al., Eds., Handbook ofPharmaceutical Excipients, American Pharmaceutical Association,Washington.

The term “prodrug” refers to a compound that is metabolized or otherwiseconverted to a biologically active or more active compound or drug,typically after administration. A prodrug, relative to the drug, ismodified chemically in a manner that renders it, relative to the drug,less active, essentially inactive, or inactive. However, the chemicalmodification is such that the corresponding drug is generated bymetabolic or other biological processes, typically after the prodrug isadministered.

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc. In aparticular embodiment, the treatment of a retroviral infection resultsin at least an inhibition/reduction in the number of infected cellsand/or detectable viral levels.

As used herein, the term “prevent” refers to the prophylactic treatmentof a subject who is at risk of developing a condition (e.g., HIVinfection) resulting in a decrease in the probability that the subjectwill develop the condition.

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease. The treatmentof a microbial infection (e.g., HIV infection) herein may refer tocuring, relieving, and/or preventing the microbial infection, thesymptom(s) of it, or the predisposition towards it.

As used herein, the term “therapeutic agent” refers to a chemicalcompound or biological molecule including, without limitation, nucleicacids, peptides, proteins, and antibodies that can be used to treat acondition, disease, or disorder or reduce the symptoms of the condition,disease, or disorder.

As used herein, the term “small molecule” refers to a substance orcompound that has a relatively low molecular weight (e.g., less than4,000, less than 2,000, particularly less than 1 kDa or 800 Da).Typically, small molecules are organic, but to are not proteins,polypeptides, or nucleic acids, though they may be amino acids ordipeptides.

The term “antimicrobials” as used herein indicates a substance thatkills or inhibits the growth of microorganisms such as bacteria, fungi,viruses, or protozoans.

As used herein, the term “antiviral” refers to a substance that destroysa virus and/or suppresses replication (reproduction) of the virus. Forexample, an antiviral may inhibit and or prevent: production of viralparticles, maturation of viral particles, viral attachment, viral uptakeinto cells, viral assembly, viral release/budding, viral integration,etc.

As used herein, the term “highly active antiretroviral therapy” (HAART)refers to HIV therapy with various combinations of therapeutics such asnucleoside reverse transcriptase inhibitors, non-nucleoside reversetranscriptase inhibitors, HIV protease inhibitors, and fusioninhibitors.

As used herein, the term “amphiphilic” means the ability to dissolve inboth water and lipids/apolar environments. Typically, an amphiphiliccompound comprises a hydrophilic portion and a hydrophobic portion.“Hydrophobic” designates a preference for apolar environments (e.g., ahydrophobic substance or moiety is more readily dissolved in or wettedby non-polar solvents, such as hydrocarbons, than by water).“Hydrophobic” compounds are, for the most part, insoluble in water. Asused herein, the term “hydrophilic” means the ability to dissolve inwater.

As used herein, the term “polymer” denotes molecules formed from thechemical union of two or more repeating units or monomers. The term“block copolymer” most simply refers to conjugates of at least twodifferent polymer segments, wherein each polymer segment comprises twoor more adjacent units of the same kind.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof (e.g., scFv), that binds to a specificantigen. As used herein, antibody or antibody molecule contemplatesintact immunoglobulin molecules, immunologically active portions of animmunoglobulin molecule, and fusions of immunologically active portionsof an immunoglobulin molecule.

As used herein, the term “immunologically specific” refers toproteins/polypeptides, particularly antibodies, that bind to one or moreepitopes of a protein or compound of interest, but which do notsubstantially recognize and bind other molecules in a sample containinga mixed population of antigenic biological molecules.

As used herein, the term “targeting ligand” refers to any compound whichspecifically binds to a specific type of tissue or cell type,particularly without substantially binding other types of tissues orcell types. Examples of targeting ligands include, without limitation:proteins, polypeptides, peptides, antibodies, antibody fragments,hormones, ligands, carbohydrates, steroids, nucleic acid molecules, andpolynucleotides.

The term “aliphatic” refers to a non-aromatic hydrocarbon-based moiety.Aliphatic compounds can be acyclic (e.g., linear or branched) or cyclicmoieties (e.g., cycloalkyl) and can be saturated or unsaturated (e.g.,alkyl, alkenyl, and alkynyl). Aliphatic compounds may comprise a mostlycarbon main chain (e.g., 1 to about 30 carbons) and comprise heteroatomsand/or substituents (see below). The term “alkyl,” as employed herein,includes saturated or unsaturated, straight or branched chainhydrocarbons containing 1 to about 30 carbons in the normal/main chain.The hydrocarbon chain of the alkyl groups may be interrupted with one ormore heteroatom (e.g., oxygen, nitrogen, or sulfur). An alkyl (oraliphatic) may, optionally, be substituted (e.g. with fewer than about8, fewer than about 6, or 1 to about 4 substituents). The term “loweralkyl” or “lower aliphatic” refers to an alkyl or aliphatic,respectively, which contains 1 to 3 carbons in the hydrocarbon chain.Alkyl or aliphatic substituents include, without limitation, alkyl(e.g., lower alkyl), alkenyl, halo (such as F, Cl, Br, I), haloalkyl(e.g., CCl₃ or CF₃), alkoxyl, alkylthio, hydroxy, methoxy, carboxyl,oxo, epoxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl (e.g.,NH₂C(═O)— or NHRC(═O)—, wherein R is an alkyl), urea (—NHCONH₂),alkylurea, aryl, ether, ester, thioester, nitrile, nitro, amide,carbonyl, carboxylate and thiol. Aliphatic and alkyl groups having atleast about 5 carbons in the main chain are generally hydrophobic,absent extensive substitutions with hydrophilic substituents.

The term “aryl,” as employed herein, refers to monocyclic and bicyclicaromatic groups containing 6 to 10 carbons in the ring portion. Examplesof aryl groups include, without limitation, phenyl or naphthyl, such as1-naphthyl and 2-naphthyl, or indenyl. Aryl groups may optionallyinclude one to three additional rings fused to a cycloalkyl ring or aheterocyclic ring. Aryl groups may be optionally substituted throughavailable carbon atoms with, for example, 1, 2, or 3 groups selectedfrom hydrogen, halo, alkyl, polyhaloalkyl, alkoxy, alkenyl,trifluoromethyl, trifluoromethoxy, alkynyl, aryl, heterocyclo, aralkyl,aryloxy, aryloxyalkyl, aralkoxy, arylthio, arylazo, heterocyclooxy,hydroxy, nitro, cyano, sulfonyl anion, amino, or substituted amino. Thearyl group may be a heteroaryl. “Heteroaryl” refers to an optionallysubstituted, mono-, di-, tri-, or other multicyclic aromatic ring systemthat includes at least one, and preferably from 1 to about 4, sulfur,oxygen, or nitrogen heteroatom ring members. Heteroaryl groups can have,for example, from about 3 to about 50 carbon atoms (and all combinationsand subcombinations of ranges and specific numbers of carbon atomstherein), with from about 4 to about 10 carbons being preferred.

The following examples provide illustrative methods of practicing theinstant invention and are not intended to limit the scope of theinvention in any way.

Example 1

Maximal reduction of residual HIV-1 from its tissue sanctuary sites thatinclude brain, lymph nodes, bone marrow, gut-associated lymphoid tissueand the genital tracts can be achieved by development of long actingreservoir targeted medicines. In addition to the benefit of infrequentdosing intervals, long acting injectable drug formulations can bedesigned to utilize receptor mediated processes to achieve improved celltargeting, extended drug half-life, and enhanced tissue biodistribution.CAB is a potent viral integrase inhibitor and has been formulated as aLAP (CAB-LAP) which demonstrates sustained plasma drug levels in humansafter single intramuscular dose. Long-acting injectable nanoformulationsof rilpivirine and CAB-LAP have already enabled once-monthly injectionfor HIV suppression and prevention (Andrews, et al. (2014) Science343(6175):1151-1154; Cohen, J. (2014) Science 343(6175):1067; Spreen, etal. (2013) Curr. Opin. HIV AIDS, 8(6):565-571). The main limitations ofexisting nanoformulations include requirement for high doses and highinjection volumes. To this end, long acting slow effective releaseantiretroviral therapies (LASER ART) have been developed by synthesizinglipophilic and hydrophobic prodrug nanocrystals that permit rapid drugpenetration across physiological barriers (Lin, et al. (2018) Chem.Commun. (Camb) 54:8371-4; Montenegro-Burke, et al. (2018) JR, Woldstad CJ, Fang M, Bade A N, McMillan J, Edagwa B, et al. (2018) Mol.Neurobiol., 56(4):2896-2907; Thomas, et al., (2018) M B, Gnanadhas D P,Dash P K, Machhi J, Lin Z, McMillan J, et al. (2018) Nanomedicine (Lond)13(17):2139-2154; Gu, et al. (2018) PLoS Pathog., 14:e1007061; Zhou, etal. (2018) Biomaterials 151:53-65; Kevadiya, et al. (2018) Theranostics8:256-76; Sillman, et al. (2018) Nat. Commun., 9:443; McMillan, et al.(2018) Antimicrob. Agents Chemother., 62: e01316-17; Gnanadhas, et al.(2017) J. Clin. Invest., 127:857-73). LASER ART maximizes drug loadingwith limited excipient usage, while maintaining scalability andlong-term storage. Myristoylated prodrugs have been formulated withpoloxamer surfactants. Improved potency, bioavailability, and tissuedistribution of CAB was demonstrated by increasing drug lipophilicitythat sustained plasma CAB concentrations at the PA-IC₉₀ for 4 months inrhesus macaques after single 45 mg/kg CAB equivalent intramuscularinjection. Here, improved prodrugs and nanoformulations have beensynthesized which reduce dosing frequency while improving viralreservoir targeting and drug activity.

Fatty diester and monoester integrase inhibitor prodrugs are providedherein. Fatty ester CAB dimers and substitution with amino acid fattyesters can extend the injecting dosing intervals from >6 months to ayear. These integrase inhibitor formulations will reduce injectionvolumes and readily cross-cell and tissue barriers. Optimal use oflipophilic drug nanocrystals will facilitate macrophage and CD4+ T cellparticle uptake. Integrase inhibitors will be modified, for example,using azeloyl and steroyl diacids, alanyl and phenylalanyl palmitoylesters. Lipophilic prodrugs will enhance intracellular delivery of theintegrase inhibitor and improve potency. One means of decreasinginjection volumes is through positively affecting drug potency. Theproposed ester derivatizing promoieties are biocompatible and as suchadjust to their microenvironment (Remenar, J. F. (2014) Mol. Pharm.,11:1739-49). The hydrocarbon chain length will tightly control prodrugbioconversion, while hydrophobic amino acid side chains improve drug topolymer interactions affecting formulation stability and intracellularprodrug hydrolysis (Remenar, J. F. (2014) Mol. Pharm., 11:1739-49;Ikuta, et al. (2015) Chem. Commun. (Camb) 51:12835-8). Abilities tocontrol drug release by changing dimer design allows drug release ratesand activation to be tuned depending on the prodrug and particle. Slowprodrug conversion in blood also allows the drug to redistribute intotissue rather than causing spikes and troughs in active CABconcentration. Amino acid modified esters not only improve prodrug andformulation stability but have also facilitate active transport ofactive compounds across cell membranes (Vig, et al. (2003) Pharm. Res.,20:1381-8). Cell and tissue targeted formulations will optimize drugentry and retention. Dissolution rates of drug and subsequent diffusioninto extracellular media will control rate of release and prodrugbioconversion.

Fatty diester CAB (M3CAB and M4CAB) and amino acid modified fatty esterCAB prodrugs (M5CAB and M6CAB) will be synthesized using a one-stepscalable synthesis scheme (Zhou, et al. (2018) Biomaterials 151:53-65;Sillman, et al. (2018) Nat. Commun., 9:443). Briefly, the parent drugwill be dissolved in dimethylformamide, followed by deprotonation withN,N-diisopropyethylamine base and conjugation to fatty acyl chloridesunder an inert atmosphere (FIG. 1).

Prodrug chemical structures and crystallinity will be evaluated bynuclear magnetic resonance (NMR), Fourier-transform infraredspectroscopy (FTIR), mass spectrometry (MS), and X-ray diffraction(XRD). Prodrug potency is linked to cleavage of derivatizing promoieties(Birkus, et al. (2008) Mol. Pharmacol., 74:92-100; Birkus, et al. (2007)Antimicrob. Agents Chemother., 51:543-50; Feng, et al. (2018)Antimicrob. Agents Chemother., 62: e00620-18; Okon, et al. (2017) ACSMed. Chem. Lett., 8:958-62; McGuigan, et al. (2010) J. Med. Chem.,53:4949-57). Enzymatic and chemical stability studies will be performedto determine prodrug hydrolysis, metabolic activation and efficacy. Thecompounds will be dissolved in sera of multiple species and buffersfollowed by prodrug and active drug quantitation by mass spectrometry.Prodrug metabolism in liver homogenates and hepatocytes from rat,rabbit, dog and human will be determined (Hoppe, et al. (2014) J. Pharm.Sci., 103:1504-14). Intracellular activation of the synthesized prodrugsin subcellular compartments in the presence and absence of variouscathepsin and peptidase inhibitors will also be determined. The role ofhydrolytic enzymes in ester bond cleavage will be assessed in efficacystudies that will include determination of EC₅₀ of each compound in MDMand CD4+ T cells. Prodrugs will then proceed to formulation-screeningphase where compound solubility profile, thermal and chemical propertieswill be evaluated to establish excipient compatibility and potentialdrug release profiles. CAB apparent half-life will be extended bytransformation into lipophilic CAB prodrugs. Prodrug formation is at thecore of strategy reflecting masked forms of native drugs that becomeactivated through enzymatic and/or chemical hydrolysis with no change inpharmacological outcomes (Huttunen, et al. (2011) Pharmacol. Rev.,63:750-71; Anastasi, et al. (2003) Curr. Med. Chem., 10:1825-43).Prodrugs offer therapeutic benefits over native compounds with reducedpre-systemic metabolism and toxicity and increased lipophilicity forenhanced cell membrane and tissue permeability (Rautio, et al. (2008)Nat. Rev. Drug Discov., 7:255-70; Park, et al. (2013) Arch. Pharm. Res.,36:651-9).

Particle uptake, retention, release and sustained antiretroviralactivities in both MDM and CD4+ T cells will also be evaluated. First,MDM will be treated with the prodrug formulations. These will be testedfor uptake (2 to 24 hours), retention, release, and antiretroviralactivities (1 to 30 days). Drug content in MDM will be quantified byhigh performance liquid chromatography (HPLC). Antiretroviral drugactivity tests will be performed by measures of RT activity andcell-associated HIV-1 p24 in cells previously challenged with virus (ADAor other CCR5 strains such as DJV and Yu2). Cells will first be givenprodrug formulations then challenged with virus from one to 30 days.Native drug formulation treatments will serve as controls.

Antiretroviral, immune, and neuroprotective outcomes in humanized micewill be assessed at the tissue and cellular levels. Immune deficientmice transplanted with CD34+ hematopoietic stem cells will be used toassess whether nanoformulations could reach sanctuary sites to protectmice from infection (Gorantla, et al. (2012) J. Neuroimmune Pharmacol.,7(2):352-62; Gorantla, et al. (2010) Am. J. Pathol., 177:2938-49; Dash,et al. (2011) J. Neurosci., 31:3148-57; Gorantla, et al. (2010) J.Immunol., 184:7082-91; Gorantla, et al. (2007) J. Virol., 81:2700-12).Humanized mice will be administered a single IM dose of ARV formulationsat a concentration of 45 mg/kg at 22 weeks of age. Mice will bechallenged two weeks post drug treatment with 2×10⁴ TCID₅₀ ofHIV-1_(ADA) by IP injection. Plasma will be used for quantitative viralload measurements. PrEP tests will be performed in mice challenged up toone year. Following study termination, spleen, thymus, lymph nodes,liver, lungs, gut, genitourinary tissues, and brain will be collected at3, 6, 9 and 12 months for drug quantitation and cell profiling. HIV-1p24staining, nested and droplet digital PCR, DNA and RNA scope, and viralload will be measured.

Example 2

The DTG prodrugs M3DTG and M4DTG were studied. First, antiretroviralefficacy was determined by measurement of HIV reverse transcriptase (RT)activity. To assess antiretroviral efficacy, monocyte-derived macrophage(MDM) were treated with either M3DTG or M4DTG for 8 hours over a rangeof concentrations (0.01-1000 nM). Drug was dosed in 0.1% (v/v) DMSO inmedia. After treatment, cells were washed with PBS to remove excess offree drug and nanoparticles and the cells were cultured with freshmedia, with half-media exchanges every other day. The MDM werechallenged with HIV-1_(ADA) at a MOI of 0.01 infectious viralparticles/cell for up to 30 days. Progeny virion production was measuredby RT activity in culture medium (Kalter, et al. (1992) J. Clin.Microbiol., 30(4):993-995). As seen in FIG. 2A, both M3DTG and M4DTGpossess significant antiretroviral activity.

Nanoformulations were generated as follows. Drug nanocrystals werecoated with poloxamer 407 (P407). The nanocrystals may also bestabilized with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000 (DSPE-PEG), and/or polyvinyl alcohol (PVA), polysorbateand/or polyethylene glycol surfactants. Briefly, drug and P407 weremixed in endotoxin free water. The premixed suspensions were formulatedby wet milling or high-pressure homogenization at 20,000 psi pressureuntil desirable size and polydispersity index (PDI) were achieved.Nanoformulations were characterized for particle size, polydispersityindex (PDI) and zeta potential by dynamic light scattering (Table 1).This was done using a Malvern Zetasizer, Nano Series Nano-ZS (MalvernInstruments Inc, Westborough, Mass.). Nanoparticle morphology wasdetermined by scanning electron microscopy (SEM). UPLC MS/MS was usedfor drug quantitation.

TABLE 1 Formulation characterization. Drug: Size Zeta Formulation BufferPolymer Purification (nm) PDI (mV) NDTG Water 2:1 Centrifugation 3580.25  −8.6 NM3DTG 10 mM 2:1 Centrifugation 277 0.22 −34.3 HEPES, pH 7.8NM4DTG 10 mM 2:1 Centrifugation 370 0.32 −31.8 HEPES, pH 7.8

Cell vitality was also assessed. Briefly, cellular viability followingtreatment with nanoparticles was evaluated by performing a3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. Human MDM plated in 96-well plates at a density of 0.08×10⁶ cellsper well were treated with various concentrations of nanoparticles for24 hours. Untreated cells were used as controls. For each group sampleswere in quadruplets. Cells were washed with PBS and incubated with 100μL/well of MTT solution (5 mg/mL) for 45 minutes at 37° C. Afterincubation, MTT solution was removed, and cells were washed with PBS.Then, 200 μL of DMSO was added to each well, and absorbance was measuredat 490 nm on a Molecular Devices SpectraMax® M3 plate reader withSoftMax Pro 6.2 software (Sunnyvale, Calif.). As seen in FIG. 2B,neither NM3DTG nor NM4DTG were toxic to the MDM cells.

Drug uptake and retention were also measured. Human monocytes wereplated in a 12-well plate at a density of 1.0×10⁶ cells per well usingDMEM supplemented with 10% heat-inactivated pooled human serum, 1%glutamine, 10 μg/mL ciprofloxacin, and 50 μg/mL gentamicin. Cells weremaintained at 37° C. in a 5% CO₂ incubator. After 7-10 days ofdifferentiation in the presence of 1000 U/mL recombinant humanmacrophage colony stimulating factor (MCSF), MDM were treated with arange of nanoformulations. Uptake of drug was assessed by measurementsof intracellular drug concentrations at various timepoints aftertreatment. For drug retention studies, cells were treated for 8 hoursthen washed with PBS and maintained with half-media changes every otherday until collection at various timepoints. For both studies, adherentMDM were washed with PBS (3×1 mL), then scraped into 1 mL of fresh PBS,and counted at indicated time points using a Countess™ automated cellcounter (Invitrogen, Carlsbad, Calif.). Cells were pelleted bycentrifugation at 4° C. The cell pellet was reconstituted in highperformance liquid chromatography (HPLC)-grade methanol and probesonicated followed by centrifugation. The supernatant was analyzed fordrug content using HPLC. DTG levels were measured for NDTG treatment,while prodrug levels were measured for prodrug formulation treatments.

As seen in FIG. 3, NM3DTG and NM4DTG were taken up by MDM todramatically higher levels than NDTG. Moreover, MDM retained NM3DTG andNM4DTG at dramatically higher levels for longer periods of time thanNDTG (FIG. 4).

Antiretroviral efficacy was also determined in MDM by HIV-1 RT activityup to 30 days after drug treatment (FIG. 5). Cells were pretreated for 8hours with equal drug concentrations of 1 μM or 10 μM NM3DTG or NM4DTG.At the indicated times cells were challenged with HIV-1_(ADA) and mediawas collected after an additional 10 days and assayed for HIV-1 RTactivity. As seen in FIG. 5, NM3DTG and NM4DTG possess significantantiretroviral activity.

Pharmacokinetics (PK) studies in mice were also performed. BALB/cJ.(Male, 6-8 weeks, Jackson Labs) were administered 45 mg/kgDTG-equivalents of NDTG, NM3DTG, or NM4DTG by a single intramuscular(IM, caudal thigh muscle) injection. Following injection, blood sampleswere collected into heparinized tubes at day 1 post-administration andthen weekly by cheek puncture (submandibular vein, MEDIpoint, Inc.,Mineola, N.Y.). Collected blood (25 μL) was immediately diluted into 1mL ACN and stored at −80° C. until drug measurements. Remaining bloodsamples were centrifuged at 2,000 g for 8 minutes for plasma collection.Plasma was collected and stored at −80° C. for analysis of drugcontents. DTG, M3DTG, and M4DTG were quantitated in mouse plasma byUPLC-MS/MS using a Waters ACQUITY H-class UPLC (Waters, Milford, Mass.,USA) connected to a Xevo TQ-S micro mass spectrometer. All solvents forsample processing and UPLC-MS/MS analysis were LCMS-grade (Fisher). 25μL of sample was added into 1 mL acetonitrile (ACN) spiked with 10 μLinternal standard (IS). Samples were vortexed and centrifuged at17,000×g for 10 minutes at 4° C. The supernatants were collected anddried using a SpeedVac® and reconstituted in 100 μL 80% methanol; 10 μLwas injected for DTG, M3DTG, and M4DTG UPLC-MS/MS analysis. Standardcurves were prepared in blank mouse plasma/blood. Spectra were analyzedand quantified by MassLynx software version 4.1. All quantitations weredetermined using analyte peak area to internal standard peak arearatios.

As seen in FIG. 6A, all mice gained weight during the PK study,indicating good animal health. The plasma concentration of DTG orprodrug is shown in FIG. 6B. At day 1 post-injection, NDTG treatmentgenerated higher plasma DTG concentrations compared to both NM3DTG andNM4DTG and showed dramatically faster decay kinetics over the studyperiod in comparison to NM3DTG and NM4DTG. With NDTG treatment, plasmaDTG concentrations were maintained above the four times protein-adjusted90% inhibitory concentration (4×PA-IC₉₀) up to about day 20 (792.7ng/mL), then rapidly declined to below the protein associated inhibitorconcentration (PA-IC₉₀) by day 27 (75 ng/mL) before falling to near thelimit of quantitation (0.5 ng/mL) by about day 56. NM3DTG treatmentshowed significantly slower decay, and maintained plasma DTG levelsabove the 4×PA-IC₉₀ up to about day 98 and above PA-IC₉₀ for the lengthof the experiment. NM4DTG treatment also showed significantly slowerdecay, and maintained plasma DTG levels above the 4×PA-IC₉₀ up to aboutday 112 and above PA-IC₉₀ for the length of the experiment. Thus, thedata in FIG. 6B demonstrated that DTG half-life following NM3DTG orNM4DTG treatment was dramatically greater than that of NDTG.

A number of publications and patent documents are cited throughout theforegoing specification in order to describe the state of the art towhich this invention pertains. The entire disclosure of each of thesecitations is incorporated by reference herein.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1-36. (canceled)
 37. A compound, or a pharmaceutically acceptable saltthereof, comprising a first integrase inhibitor and a second integraseinhibitor, wherein said first and second integrase inhibitors arecovalently attached by a linker.
 38. The compound of claim 37, whereinsaid first and second integrase inhibitors are each independentlyselected from the group consisting of cabotegravir (CAB), raltegravir(RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir (BIC), BI224436, and MK-2048.
 39. The compound of claim 37, wherein said linkeris an optionally substituted aliphatic group, and wherein said linkerforms an ester with the oxygen of a hydroxyl moiety of said first andsecond integrase inhibitors.
 40. The compound of claim 39, wherein saidoptionally substituted aliphatic group comprises 1 to 30 carbons. 41.The compound of claim 37, wherein the compound is represented by Formula(I), Formula (II), Formula (III), Formula (IV), or Formula (V):

wherein the —(CH₂)_(n)— of Formula (I), Formula (II), Formula (III),Formula (IV), or Formula (V) is optionally substituted with at least oneheteroatom; and n is an integer from 1 to
 24. 42. The compound of claim41, wherein n is an integer from 6 to
 14. 43. The compound of claim 37,wherein the compound is


44. A compound, or a pharmaceutically acceptable salt thereof,comprising an integrase inhibitor conjugated to an amino acid fattyester, wherein the amino acid fatty ester comprises an aliphatic group.45. The compound of claim 44, wherein said integrase inhibitor isselected from the group consisting of cabotegravir (CAB), raltegravir(RAL), elvitegravir (EVG), dolutegravir (DTG), bictegravir (BIC), BI224436, and MK-2048.
 46. The compound of claim 44, wherein the compoundis represented by Formula (VI), Formula (VII), Formula (VIII), Formula(IX), or Formula (X):

wherein: R is an optionally substituted aliphatic group; and AA is oneor more amino acids.
 47. The compound of claim 46, wherein AA is oneamino acid.
 48. The compound of claim 46, wherein AA is selected fromthe group consisting of alanine, valine, phenylalanine, proline,tyrosine, and lysine.
 49. The compound of claim 46, wherein R is asaturated linear aliphatic chain of a length of 11 to 19 carbons. 50.The compound of claim 44, wherein said compound is


51. A nanoparticle, comprising a compound or a pharmaceuticallyacceptable salt thereof of claim 37, and at least one polymer orsurfactant.
 52. The nanoparticle of claim 51, wherein said polymer orsurfactant is an amphiphilic block copolymer.
 53. The nanoparticle ofclaim 52, wherein said amphiphilic block copolymer comprises at leastone block of poly(oxyethylene) and at least one block ofpoly(oxypropylene).
 54. The nanoparticle of claim 51, wherein thepolymer or surfactant is P407.
 55. A pharmaceutical composition,comprising a compound or a pharmaceutically acceptable salt thereof ofclaim 37, and at least one pharmaceutically acceptable carrier.
 56. Amethod of treating a viral infection in a subject in need thereof,comprising administering a therapeutically effective amount of apharmaceutical composition of claim 55 to the subject.
 57. The method ofclaim 56, wherein the viral infection is an HIV infection.